Integrated transcriptomic and metabolomic analyses reveal key genes controlling flavonoid biosynthesis in Citrus grandis ‘Tomentosa’ fruits

Background: As a well-recognized Traditional Chinese medicine (TCM), immature fruits of Citrus grandis ‘Tomentosa’ serve to cure chronic cough in humans, to which flavonoids may contribute. Knowledge about the molecular mechanisms underlying flavonoid biosynthesis in ‘Tomentosa’ fruits would, therefore, favor the breeding of varieties with improved health supporting properties. Results To this end, we determined the transcriptomes, using RNA-seq, and metabolic profiles of two contrasting C. grandis varieties, namely ‘Tomentosa’ (‘ZM’) used for TCM production, and a locally cultivated pomelo (‘GQ’), at four fruit development stages. A total of 39 flavonoids, including 14 flavanone/flavone, five isoflavonoids, 12 flavonols, and six anthocyanins, were identified and 16 of them were quantitatively determined in fruits of the two varieties. We found that ‘ZM’ fruits contain more flavonoids than ‘GQ’, whereby rhoifolin accumulated to significantly higher levels in ‘ZM’. Transcriptome sequencing of ‘ZM’ and ‘GQ’ allowed the annotation of 31,510 genes, including 1,387 previously unknown ones. A total of 646 genes were found to be differentially expressed between ‘ZM’ and ‘GQ’ throughout all four fruit developmental stages, indicating them as robust expression markers in future breeding programs. Weighted gene co-expression network analysis (WGCNA) identified 18 modules. Conclusions Combined transcriptional and metabolic analysis revealed 25 flavonoid biosynthesis structural genes and 16 transcriptional regulators (MYBs, bHLHs, WD40) to be likely involved in the biosynthesis of flavonoids in C. grandis ‘Tomentosa’ fruits.